Many clinicians believe that cancer is an organ-confined disease in its early stages. However, it appears that this notion is incorrect, and cancer is often a systemic disease by the time it is first detected using methods currently available. There is evidence that primary cancers begin shedding neoplastic cells into the circulation at an early disease stage prior to the appearance of clinical manifestations. Upon vascularization of a tumor, tumor cells shed into the circulation may attach and colonize at distant sites to form metastases. These circulating tumor cells (CTC) contain markers not normally found in healthy individuals' cells, thus forming the basis for diagnosis and treatment of specific carcinomas. Hence, the presence of tumor cells in the circulation can be used to screen for cancer in place of, or in conjunction with, other tests, such as mammography, or measurements of PSA. By employing appropriate monoclonal antibodies directed to associated markers on or in target cells, or by using other assays for cell protein expression, or by the analysis of cellular mRNA, the organ origin of such cells may readily be determined, e.g., breast, prostate, colon, lung, ovarian or other non-hematopoietic cancers.
Thus, in cases where cancer cells can be detected, while there are essentially no clinical signs of a tumor, it will be possible to identify their presence as well as the organ of origin. Furthermore, based on clinical data, cancer should be thought of as a blood borne disease characterized by the presence of potentially very harmful metastatic cells, and therefore, treated accordingly. In cases where there is absolutely no detectable evidence of CTC, e.g., following surgery, it may be possible to determine from further clinical study whether follow-up treatment, such as radiation, hormone therapy or chemotherapy is required. Predicting the patient's need for such treatment, or the efficacy thereof, given the costs of such therapies, is a significant and beneficial piece of clinical information. It is also clear that the number of tumor cells in the circulation is related to the stage of progression of the disease, from its inception to the final phases of disease.
Malignant tumors are characterized by their ability to invade adjacent tissue. In general, tumors with a diameter of 1 mm are vascularized and animal studies show that as much as 4% of the cells present in the tumor can be shed into the circulation in a 24 hour period (Butler, TP & Gullino PM, 1975 Cancer Research 35:512-516). The shedding capacity of a tumor is most likely dependent on the aggressiveness of the tumor. Although tumor cells are shed into the circulation on a continuous basis, it is believed that none or only a small fraction will give rise to distant metastasis (Butler & Gullino, supra). Increase in tumor mass might be expected to be proportional to an increase in the frequency of the circulating tumor cells. If this were found to be the case, methods available with a high level of sensitivity would facilitate assessment of tumor load in patients with distant metastasis as well as those with localized disease. Detection of tumor cells in peripheral blood of patients with localized disease has the potential not only to detect a tumor at an earlier stage but also to provide indications as to the potential invasiveness of the tumor.
However, whole blood is a complex body fluid containing diverse populations of cellular and soluble components capable of undergoing numerous biochemical and enzymatic reactions in vivo and in vitro, particularly on prolonged storage for more than 24 hrs. Some of these reactions are related to immunoreactive destruction of circulating tumor cells that are recognized as foreign species. The patient's immune response weakens or destroys tumor cells by the normal defense mechanisms including phagocytosis and neutrophil activation. Chemotherapy similarly is intended to reduce both cell function and proliferation by inducing cell death by necrosis. Besides these external destructive factors, tumor cells damaged in a hostile environment may undergo programmed death or apoptosis. Normal and abnormal cells (including CTC) undergoing apoptosis or necrosis, have altered membrane permeabilities that allow escape of DNA, RNA, and other intracellular components leading to formation of damaged cells, fragmented cells, cellular debris, and eventual complete disintegration. Such tumor cell debris may still bear epitopes or determinants characteristic of intact cells and can lead to spurious increases in the number of detected circulating cancer cells. Whole blood specimens from healthy individuals also have been observed to undergo destruction of labile blood cell components, herein categorized as decreased blood quality, on prolonged storage for periods of greater than 24 hours. For example, erythrocytes may rupture and release hemoglobin and produce cell ghosts. Leukocytes, particularly granulocytes, are known to be labile and diminish on storage. Such changes increase the amount of cellular debris that can interfere with the isolation and detection of rare target cells such as CTC. The combined effects of these destructive processes can substantially increase cellular debris, which is readily detectable, for instance, in flow cytometric and microscopic analyses, such as CELLSPOTTER, an image analysis device, or CELLTRACKS®, a semi-automated image analysis device, which are described in commonly-owned U.S. Pat. Nos. 5,985,153 and 6,136,182, both of which are incorporated by reference herein.
Detection of circulating tumor cells by microscopic imaging is similarly adversely affected by spurious decreases in classifiable tumor cells and a corresponding increase in interfering stainable debris. Hence, maintaining the integrity or the quality of the blood specimen is of utmost importance, since there may be a delay of as much as 24 hours between blood draw and specimen processing. Such delays are to be expected, since the techniques and equipment used in processing blood for this assay may not be readily available in every laboratory. The time necessary for a sample to arrive at a laboratory for sample processing may vary considerably. It is therefore important to establish the time window within which a sample can be processed. In routine hematology analyses, blood samples can be analyzed within 24 hours. However, as the analysis of rare blood cells is more critical, the time window in which a blood sample can be analyzed is shorter. An example is immunophenotyping of blood cells, which, in general, must be performed within 24 hours. In a cancer cell assay, larger volumes of blood have to be processed, and degradation of the blood sample can become more problematic as materials released by disintegrating cells, both from CTC and from hematopoietic cells, can increase the background and therefore decrease the ability to detect tumor cells.
The origin and nature of observed small debris and large clump-like aggregates are not fully understood, but are believed to involve cellular components or elements originating from target cells, non-target cells, and possibly plasma components. Since CTC can be considered immunologically foreign species, normal cellular immune responses of the host will occur in vivo even before blood draw. Also large numbers of CTC can be continuously shed from a tumor site, and a steady-state level is maintained in which destruction of CTC equals the shedding rate which in turn depends on the size of the tumor burden (see J G Moreno et al. “Changes in Circulating Carcinoma Cells in Patients with Metastatic Prostate Cancer Correlates with Disease State.” Urology 58. 2001).
Various methods are known in this particular art field for recovering tumor cells from blood. For example, U.S. Pat. No. 6,190,870 to AmCell and Miltenyi teaches immunomagnetic isolation followed by flow cytometric enumeration. However, before immunomagnetic separation, the blood samples are pre-processed using density gradients. Furthermore, there is no discussion of isolating or counting anything other than intact cells. There is also no visual analysis of the samples.
In U.S. Pat. No. 6,197,523, Rimm et al. describe enumerating cancer cells in 100 μl blood samples. The methods use capillary microscopy to confirm the identity of cells that are found. The methods are specific for intact cells, and there is no discussion of isolating or enumerating anything else, such as fragments or debris.
In U.S. Pat. No. 6,365,362 to Immunivest, methods are described for immunomagnetically enriching and analyzing samples for tumor cells in blood. The methods are specifically directed towards analyzing intact cells, where the number of cells correlates with the disease state. The isolated cells are labeled for the presence of nucleic acid and an additional marker, which allows the exclusion of non-target sample components during analysis.
In WO02/20825, Chen describes using an adhesion matrix for enumerating tumor cells. Briefly, the matrix is coated with specific adhesion molecules that will bind to cancer cells with metastatic potential. The matrix can then be analyzed for the presence and type of captured cells. Also described are methods for using the matrix in screening treatments. While steps are taken to discriminate between intact cells and apoptotic or necrotic cells, the apoptotic or necrotic cells are specifically excluded from analysis.
In WO00/47998, two pathways are described for CTC, terminal and proliferative. Both pathways begin with an “indeterminate” cell that progresses, as determined by morphological differences, down either the terminal or proliferative pathway. A cell in the terminal pathway eventually is destroyed, and a cell in the proliferative pathway will form a new metastatic colony as a metastatic tumor. These two pathways were designed to explain morphological differences seen in patient samples.
Generally, the more resistant and proliferative cells survive to establish secondary or metastatic sites. In the peripheral circulation, CTC are further attacked in vivo (and also in vitro) by activated neutrophils and macrophages resulting progressively in membrane perforation, leakage of electrolytes, smaller molecules, and eventual loss of critical cellular elements including DNA, chromatin, etc, which are essential for cell viability. At a critical point of the cell's demise, cell destruction is further assisted by apoptosis. Apoptosis is characterized by a series of stepwise slow intracellular events, which differs from necrosis or rapid cell death triggered or mediated by an extracellular species, e.g. a cytotoxic anti-tumor drug. All or some of these destructive processes may lead to formation of debris and/or aggregates including stainable DNA, DNA fragments and “DNA ladder” structures from disintegrating CTC as well as from inadvertent destruction of normal hematopoietic cells during drug therapy, since most cytotoxic drugs are administered at near toxic doses.
As shown in WO00/47998, U.S. Pat. No. 6,190,870, and other publications, CTC can circulate as both live and dead cells, wherein “dead” comprises the full range of damaged and fragmented cells as well as CTC-derived debris. The tumor burden is probably best represented by the total of both intact CTC and of damaged CTC, which bear morphological characteristics of cells. However, some damaged cells, may have large pores allowing leakage of the liquid and particulate cytosolic contents resulting in a change in the buoyant densities from about 1.06-1.08 to greater than 1.12, or well above the densities of RBC (live and dead cells can be separated at the interface of gradients of d=1.12 and 1.16 according to a Pharmacia protocol). Conventional density gradients, as used in WO00/47998 would lose such damaged CTC in the discarded RBC layer having a range in density of about 1.08 to 1.11. CTC debris that is positively stained for cytokeratin may also have densities falling in the RBC or higher ranges, since most intracellular components (with the possible exception of lipophilic membrane fragments) have densities in the range of 1.15 to 1.3. Hence, a substantial portion of damaged CTC and CTC debris may be located in or below the RBC layer, and would not be seen by the density gradient methods in WO00/47998. Some images of damaged or fragmented CTC are shown, but it is quite possible the damage occurred during cytospin or subsequent processing, and is thus artifactual. While the densities of most intact tumor cells may fall in the WBC region, it is quite likely that damaged CTC in patient samples have higher densities that may place them in the RBC layer; outside the reach of gradient techniques.
U.S. patent application No. 2001/0024802 describes methods for binding fragments and debris to beads. That published application described numerous possibilities for the density of fragments and debris of interest. Upon centrifugation, the beads will be located in a layer above RBC, because of the pre-determined specific gravity (density) of the beads coupled to fragments and/or debris. However, this system is dependent on correctly binding fragments and debris to these beads. If any other sample component binds the beads, they may not appear in the desired location, and subsequently will not be subject to analysis.
Epithelial cells in their tissue of origin obey established growth and development “rules”. Those rules include population control. This means that under normal circumstances the number and size of the cells remains constant and changes only when necessary for normal growth and development of the organism. Only the basal cells of the epithelium or immortal cells will divide and they will do so when it is necessary for the epithelium to perform its function, whatever it is depending in the nature and location of the epithelium. Under some abnormal but benign circumstances, cells will proliferate and the basal layer will divide more than usual, causing hyperplasia. Under some other abnormal but benign circumstances, cells may increase in size beyond what is normal for the particular tissue, causing cell gigantism, as in folic acid deficiency.
Epithelial tissue may increase in size or number of cells also due to pre-malignant or malignant lesions. In these cases, changes similar to those described above are accompanied by nuclear abnormalities ranging from mild in low-grade intraepithelial lesions to severe in malignancies. It is believed that changes in these cells may affect portions of the thickness of the epithelium and as they increase in severity will comprise a thicker portion of such epithelium. These cells do not obey restrictions of contact inhibition and continue growing without tissue controls. When the entire thickness of the epithelium is affected by malignant changes, the condition is recognized as a carcinoma in situ (CIS).
The malignant cells eventually are able to pass through the basement membrane and invade the stroma of the organ as their malignant potential increases. After invading the stroma, these cells are believed to have the potential for reaching the blood vessels. Once they infiltrate the blood vessels, the malignant cells find themselves in a completely different environment from the one they originated from.
The cells may infiltrate the blood vessels as single cells or as clumps of two or more cells. A single cell of epithelial origin circulating through the circulatory system is destined to have one of two outcomes. It may die or it may survive.
Single Cells:
The cell may die either through apoptosis due to internal changes or messages in the cell itself. These messages may have been in the cell before intravasation or they may be received while in the blood, or it may die due to the influence of the immune system of the host, which may recognize these cells as “alien” to this environment. The results of cellular death are identifiable in CELLSPOTTER, an image analysis device. as enucleated cells, speckled cells or amorphous cells. These cells do not have the potential for cell division or for establishing colonies or metastases.                Enucleated cells are the result of nuclear disintegration and elimination-karyorrhexis and karyolysis. They are positive for cytokeratin, and negative for nucleic acid.        The speckled cells are positive for cytokeratin and DAPI and show evidence of cellular degeneration and cytoplasmic disintegration. These cells may represent response to therapy or to the host's immune system as the cytoskeletal proteins retract.        Another dying tumor cell identifiable using CELLSPOTTER, an image analysis device, is the amorphous cell. These cells are probably damaged during the preparation process, a sign that these may be weaker, more delicate cells but may also be the result of apoptosis or immune attack.        
A single epithelial malignant cell may have the potential to survive the circulation and form colonies in distant organs. These “survivor cells” appear in CELLSPOTTER, an image analysis device, as intact cells with high nuclear material/cytoplasmic material ratio. These cells are probably undifferentiated and can potentially divide in blood and form small clumps that may extravasate in a distant capillary, where the cell may establish a new colony, or it may remain as a single cell until it extravasates, dividing once it establishes itself in the new tissue, starting this way a new colony.
Clusters: The primary tumor may shed clusters that enter the circulation as described by B Brandt et al. (“Isolation of prostate-derived single cells and cell clusters from human peripheral blood.” Cancer Research 56 p4556-4561, 1996). These clusters may remain as clusters and invade a distant tissue or they may become dissociated in the circulation, probably due to differences in pressure in blood or to the immune system's intervention. If these cells are dissociated into single cells, they may follow one of the two paths described for single cells above (see 1 and 2). Cluster formations may have an effect in survival by using the outside cells as a shield that protects the inner cells from the immune system.
Once a new colony is established in a new organ, some malignant cells will continue replicating to form a new tumor. If they reach new capillaries, the metastasis story may be repeated and a secondary metastasis occurs.
Monitoring of treatment in patients with known carcinomas: A decrease in the number of tumor cells and/or increase in the response index may represent a response to patient therapy.
                Total tumor cells=Dying cells+Survivor cells (TTC=DC+SC)        Response Index=dying cells/total tumor cells (RI=DC/TTC).        
The higher the response index, the better the response to therapy. A low response index may indicate that the patient is not responding to the treatment and or that the pt's immune system is not able to handle the tumor load.
A patient who has 50 total tumor cells that were all survivor cells at pre-treatment visit (a RI=0/50=0) and has 50 TTC on follow-up (after treatment) visit may have different outcomes depending in the RI. If all the TTC are SC (i.e. DC=0), there was no response to therapy. If there are 50 cells but the response index is 40/50=0.8, then either the immune system or the therapy is having a negative effect on tumor load, therefore, is a positive response.
Decisions in follow-up on patients with known pre-malignancies: When a pap smear is diagnosed as having cells with atypical or low-grade intraepithelial lesions, there is always the possibility that these patients have a more severe abnormality, which cells were missed as a sampling error. These patients can be colposcoped and biopsied or they may be asked to return in three months for a repeat pap smear. If the atypical cells were concurrent with a small focal area of malignant cells that did not get sampled, the patient will wait 3 months before she gets any follow-up. This may explain why some pre-malignancies seem to progress quicker than others (misdiagnoses due to sampling error, causing an artifact in statistics). These are usually explained as being a more “aggressive” pre-malignancy. CELLSPOTTER, an image analysis device, can be used to help in the decision tree of these patients. All patients with an abnormal pap (5-10% of the pap smears in the USA) can immediately be tested for circulating epithelial cells. Patients with positive tests should be followed-up immediately and aggressively. Patients with negative results may wait the three months for the repeat pap. This would simplify the decision making process for the physician and health professionals and help the patient trust her follow-up procedure.Screening: CELLSPOTTER, an image analysis device, image analysis may be used for screening of the general population with the condition that special, tissue specific antibodies would be used on a second test on all abnormal samples. Identification of CTC in a patient may indicate that there is a primary malignancy that has started or is starting the process of metastasis. If these cells are identified as of the tissue of origin with new markers, then organ specific tests, like CT guided fine needle aspirations (FNA) can be used to verify the presence or absence of such malignancies. Patients where a primary cannot be identified may be followed-up with repeat tests after establishing an individual base line.
In summary, all or some of the above-cited factors can and were found to contribute to debris and/or aggregate formation that have been observed to confound the detection of CTC by direct enrichment procedures from whole blood as disclosed in this invention. The number of intact CTC, damaged or suspect CTC as well as the degree of damage to the CTC, may further serve as diagnostically important indicators of the tumor burden, the proliferative potential of the tumor cells and/or the effectiveness of therapy. In contrast, the methods and protocols of the prior art combine unavoidable in vivo damage to CTC with avoidable in vitro storage and processing damage, thus yielding erroneous information on CTC and tumor burdens in cancer patients. Finally, the relatively simple blood test of the present invention described herein, which functions with a high degree of sensitivity and specificity, the test can be thought of as a “whole body biopsy.”